We aim to elucidate the molecular mechanisms that control the assembly of viruses with the ultimate goals of defining targets for antiviral compounds, characterizing viral antigenicity, and establishing precedents for understanding the assembly of macromolecular complexes in general. Our research focuses on the large-scale conformational changes that accompany capsid maturation and on the interaction of viruses with host cells. We are currently pursuing five subprojects. (1) Hepatitis B Virus Capsid Assembly. The HBV capsid protein presents both core antigen (capsids) and e-antigen (unassembled protein) epitopes. After first showing that capsid protein self-assembles from dimers into capsids of two different sizes, in 1997 we calculated a cryo-EM density map in which much of the secondary structure was visible, including the 4-helix bundle that forms the dimerization motif. Our subsequent research helped delineate the path of the polypeptide chain. We are now investigating the antigenic diversity of HBV capsids and the basis for e-antigenicity. New results: We characterized the epitopes of two monoclonal antibodies, including that of the first IgM to be analyzed in this way (i.e. cryo-EM and modelling). One of them differs from those of the four mAbs previously mapped, but the other is our first duplicate epitope. The two mAbs that share the same epitope have differing binding affinities and bind to capsids in differing orientations. Both observations point to differing configurations of complementarity-determining loops. Our analysis implies that the number of distinct epitopes presented on cAg capsids is of the order of twenty. That the large majority of cAg epitopes are conformational (five out of six, to date) reflects the large size of a Fab fragment and the small size of externally exposed capsid protein motifs. (2) Structure, Assembly, and Maturation of Herpesviruses. constitute an extensive family of large complex DNA viruses. Eight herpesviruses cause diseases in humans, including skin diseases. In studying herpesvirus capsid assembly, we first defined its molecular anatomy and then characterized the roles of its six major proteins. The behavior of this system is distinct from that of any other animal virus but closely resembles those of DNA bacteriophages. One major finding was our discovery of the HSV procapsid - a precursor that differs radically from the mature capsid in structure and stability. Maturation of the procapsid. controlled by the viral protease, is a target for antiviral drugs. In FY05, we analyzed maturation of the herpes simplex virus procapsid in detail, building on our previous results from time-lapse cryo-EM. This transition involves large conformational changes. The availability of a crystal structure for the protrusion domain of the major capsid protein provided an opportunity to characterize the dynamics of this event. A special computer program was written to facilitate fast and efficient fitting. Initially, there is no lateral contact between neighboring protrusion domains; ultimately, they form close contacts adding to the stability of the capsid. Our analysis monitors the dynamics of their formation. In a second line of investigation, we are applying cryo-electron tomgraphy to investigate the herpesvirus system. Progress has been limited by an inability to assess the resolution achieved in our tomograms. We derived a new resolution criterion based on a cross-validation approach. The criterion was found to be consistent with the value of resolution obtained when the tomogram happens to contain an object whose structure is known, allowing for cross-calibration. A software package for the new criterion has been written, tested, and implemented. (3) Assembly and Maturation of Bacteriophage Capsids. Our primary interest lies in the massive conformational changes that accompany capsid maturation. These transitions afford unique opportunities for insight into the regulation of protein complexes. We study maturation of several phages to exploit expedient aspects of each. New Results: (i) To investigate the thermodynamic basis of the structural changes that control HK97 maturation, we combined calorimetry with cryo-EM and found that capsid stabilization is effected solely by formation of the crosslinks, which build up progressively according to a "molecular ratchet" mechanism. Assembly of capsomers increases their melting temperature by 20 degrees. The wide denaturation endotherm of the procapsid reflects disruption of many interactions that have similar but not equal resistance to thermal perturbation. (ii) Using X-ray crystallography, we found that the minor capsid protein of T4 has the same fold as that previously determined for HK97, in the absence of sequence similarity, but also has a 60-residue insertion domain. The major T4 capsid protein should have the same fold. The emerging picture is that all five capsid proteins now solved at high or moderately high resolution have the same fold, despite minimal sequence similarity. Accordingly, it appears that most tailed phages and herpesviruses evolved from a common ancestor. (iii) We investigated the packing of DNA in the T4 head by cryo-EM, observing isometric and prolate heads in sideview and axial view. To interpret the resulting images, we performed computer modeling. We conclude that in isometric T4 heads, the DNA is spooled around the portal axis, as in T7; in prolate heads, the spool has elliptical gyres and is no longer coaxial. At the center of the head, there is a domain of less ordered DNA. These configurations represent an energy-minimized compromise between electrostatic repulsion effects between segments of DNA duplex and between DNA and the capsid wall, and the bending energy associated with coiling. (iv) Interaction of Poliovirus with Host Cells. Lacking a lipoprotein envelope, the poliovirus capsid must interact directly with the host cell membrane to effect entry and initiate infection. Previously we characterized two aspects of this interaction: (i) binding of poliovirus to its receptor; and (ii) the structural transition from the 160S state to the 135S state that follows receptor interaction. This transition involves rigid-body shifts of the cores of capsid proteins VP1, VP2, and VP3, accompanied by externalization of VP4 and the N-terminus of VP1. New results. In FY05, we extended the resolution of our cryo-EM reconstructions of 135S from 2.2 nm to 1.0 nm, allowing the beta-barrels, loops, and terminal extensions to be docked into the map with high precision. We find that VP1-N exits the capsid shell though an opening in the interface between VP1 and VP3, and the externalized N terminus of VP1 is located near the tips of propeller-like features that surround the threefold axes. (v) Papillomavirus Structure, Maturation and Antigenicity. Current vaccine trials intended to confer protection against cervical cancer utilize antibodies against the major capsid protein L1 of human papillomavirus 16 (HPV-16). However, there are some 17 cancer-causing strains of HPV, and anti-L1 antibodies are not cross-reactive against other strains. New results: In FY05, we reactivated a LSBR project that aimed to investigate the molecular anatomy of papillomaviruses. We have three goals: to localize the minor capsid protein L2; to localize the epitope of an anti-L2 antibody that appears to be cross-reactive; and to investigate size variations in our preparations of HPV virions. Cryo-EM difference imaging demonstrates that L2 is mostly sequestered under each L1 pentamer. Time-course maturation studies showed that the virion shrinks by ~ 15% as it matures. This reaction correlates with the formation of disulfide bonds.